Journal of Agricultural Technology 2016 Vol. 12(2):215-228 Available online http://www.ijat-aatsea.com ISSN 1686-9141 215 Characteristics of epididymal sperm recovered from slaughterhouse derived testes of nondescript/native goats in the Philippines Joram J. Gautane 2 , Errol Jay Y. Balagan 2 , Fely V. Manaois II 2 , Marlon B. Ocampo 1,3 , and Lerma C. Ocampo 1,2 * 1 Philippine Carabao Center-Reproductive Biotechnology Unit, Science City of Munoz, Nueva Ecija Philippines 3120 2 Central Luzon State University, College of Veterinary Science and Medicine, Science City of Munoz, Nueva Ecija Philippines 3120 3 Affilitate Faculty IGS, Central Luzon State University, Science City of Munoz, Nueva Ecija, Philippines Joram J. Gautane, Errol Jay Y. Balagan, Fely V. Manaois II, Marlon B. Ocampo, and Lerma C. Ocampo (2016) Characteristics of epididymal sperm recovered from slaughterhouse derived testes of nondescript/native goats in the Philippines. Journal of Agricultural Technology 12(2):215-228. Nondescript/ native goats are a common sight in the country side because of their ability to thrive well in harsh environmental conditions and their ability to reproduce easily despite their small size (20-30kg). In order to supply the food chain, they usually end up in the slaughterhouse shortly before or after they have reached sexual maturity. With the intensification of live importation of foreign goat breeds, there is a pressing need to conserve local native goats in the country. However native bucks are not usually trained for ejaculated semen collection therefore epididymal sperm collection from post mortem is one option. In this study, scrotal intact testes (n=6) were collected within 1-2 hour after slaughter from matured native bucks. The objective of this study is to optimize a method of sperm collection from post mortem testes and determine the sperm characteristics using basic parameters such as motility, viability, sperm concentration, and sperm volume. The slice + swim-up (Method 1) or Mince +flushing (Method 2) were performed to isolate epididymal sperm. In both methods, the obtained percentage sperm motility ranged between 60-75% by conventional method of assessment. The average sperm concentration per mL was higher in Method 2 with 1.89 ± 0.36 x10 9 sperm compared to Method 1 with 1.23 ± 0.29 x10 9 but did not differ significantly by Student’s T- test (p>0.05). In terms of sperm volume, an average of 770±200 μl was recovered in Method 2 compared with 500±110 μl in Method 1 and are not significantly different (p>0.05). In terms of percentage live sperm at the time of collection, Method 1 registered a higher mean percentage of 88.9±2.65 as compared with Method 2 with a mean percentage of 81.67±2.81 (Student’s T-test p>0.05). The proportion of normal sperm was 76.9±8.48 and 69.4±5.34 in Methods 1 and 2, respectively. The presence of proximal (16-18%) and distal droplet (69-71%) was a common find which is indicative of a sub population of maturing sperms in the epididymis. We therefore conclude that using the two recovery methods
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Journal of Agricultural Technology 2016 Vol. 12(2):215-228
Available online http://www.ijat-aatsea.com ISSN 1686-9141
215
Characteristics of epididymal sperm recovered from
slaughterhouse derived testes of nondescript/native goats in the
Philippines
Joram J. Gautane2, Errol Jay Y. Balagan
2, Fely V. Manaois II
2,
Marlon B. Ocampo1,3
, and Lerma C. Ocampo1,2
*
1Philippine Carabao Center-Reproductive Biotechnology Unit, Science City of Munoz, Nueva
Ecija Philippines 3120 2
Central Luzon State University, College of Veterinary Science and Medicine, Science City of
Munoz, Nueva Ecija Philippines 3120 3 Affilitate Faculty IGS, Central Luzon State University, Science City of Munoz, Nueva Ecija,
Philippines
Joram J. Gautane, Errol Jay Y. Balagan, Fely V. Manaois II, Marlon B. Ocampo,
and Lerma C. Ocampo (2016) Characteristics of epididymal sperm recovered from
slaughterhouse derived testes of nondescript/native goats in the Philippines. Journal of
Agricultural Technology 12(2):215-228.
Nondescript/ native goats are a common sight in the country side because of their ability to
thrive well in harsh environmental conditions and their ability to reproduce easily despite their
small size (20-30kg). In order to supply the food chain, they usually end up in the
slaughterhouse shortly before or after they have reached sexual maturity. With the
intensification of live importation of foreign goat breeds, there is a pressing need to conserve
local native goats in the country. However native bucks are not usually trained for ejaculated
semen collection therefore epididymal sperm collection from post mortem is one option. In this
study, scrotal intact testes (n=6) were collected within 1-2 hour after slaughter from matured
native bucks. The objective of this study is to optimize a method of sperm collection from post
mortem testes and determine the sperm characteristics using basic parameters such as motility,
viability, sperm concentration, and sperm volume. The slice + swim-up (Method 1) or Mince
+flushing (Method 2) were performed to isolate epididymal sperm. In both methods, the
obtained percentage sperm motility ranged between 60-75% by conventional method of
assessment. The average sperm concentration per mL was higher in Method 2 with 1.89 ±
0.36 x109 sperm compared to Method 1 with 1.23 ± 0.29 x10
9 but did not differ significantly
by Student’s T- test (p>0.05). In terms of sperm volume, an average of 770±200 µl was
recovered in Method 2 compared with 500±110 µl in Method 1 and are not significantly
different (p>0.05). In terms of percentage live sperm at the time of collection, Method 1
registered a higher mean percentage of 88.9±2.65 as compared with Method 2 with a mean
percentage of 81.67±2.81 (Student’s T-test p>0.05). The proportion of normal sperm was
76.9±8.48 and 69.4±5.34 in Methods 1 and 2, respectively. The presence of proximal (16-18%)
and distal droplet (69-71%) was a common find which is indicative of a sub population of
maturing sperms in the epididymis. We therefore conclude that using the two recovery methods
216
a considerable population of viable epididymal sperm can be isolated from post mortem gonads
of matured nondescript bucks.
Keywords: goat epididymal sperm viability and motility characteristics, post mortem testes
while dead sperms appear pink and other visible morphological abnormalities
such as coiled tail, bent tail and detached head). Some cytoplasmic droplets i.e.
white droplet in the middle part of the sperm tail can also be seen.
222
Statistical Analysis
All data gathered from the measured sperm parameters in the two
methods of sperm recovery were reported as mean ± s.e.m and significant
differences were statistically analyzed by Student’s T-test at P<0.05.
Results
In this study, testicles (n=6 pairs) from matured native goats were
immediately excised from the body after the animal was bled. Scrotal intact
native goat testicles initially measured for their weights were found to weigh
between 135-160 grams. After the scrotal sac was removed, the weight of
one testicle was between 30-40 grams averaging 37.5grams. The average
weight of the epididymis was 1.39 grams.
The volume (µL) of epididymal sperm obtained by Method 2, which is
mincing+flushing registered an average of 770 ±200 µL which was higher
but not significantly different (P>0.05) when compared with the swim-up
method (Method 2) with a mean value of 500 ±110 µL shown in table 2.
The average sperm concentration per mL ( n x10^7± s.e.m ) obtained in
method 2 was higher (189 ±36.8x10^7) compared with the swim up method
(123 ±29.7 x10^7 ). However, statistical analysis revealed no significant
difference (P>0.05) between two methods in terms sperm concentration.
Figure 5 Percentage (%) live and dead sperm, morphologically normal and
abnormal sperm in Method 1 ( blue column) and method 2 ( red column )
88.9
11.1
76.9
23.2
81.7
18.4
69.4
30.6
Live sperm Dead sperm Morphologicallynormal sperm
Morphologicallyabnormal sperm
Journal of Agricultural Technology 2016 Vol. 12(2):215-228
223
Conventional or subjective method of sperm motility evaluation
revealed sperm motility scores between 60–75% suggesting that immediately
recovered post mortem epididymal sperm remains to be alive and were still
of good quality. As shown in figure 5, a greater proportion (%) of live
sperm was obtained in Method 1 (88.9±2.76 ) compared with Method 2
(81.7±2.8) but the mean values were not statistically different by Student’s T-
test, (P>0.05). Similar to this finding is the higher percentage (% mean ±
s.e.m.) of morphologically normal sperm in method 1 (76.9 ±8.5) compared
with method 2 (69.4±5.3) but the values did not significantly differ (P>0.05).
The proportion (%) of proximal cytoplasmic droplet (16-18%) and distal
cytoplasmic droplet (69-71%) were a common find. This observation is
indicative of the presence of a sub-population of immature and maturing
sperms in the excised cauda epididymal tissue at the time of recovery. The
occurrence of other sperm abnormalities from both methods such as coiled tail
(2.2~7.6%), bent tail (4.3~5.3%) and detached head (0.4~3.1%) were also
observed and the percentage values are presented in table 3.
Table 2. Epididymal sperm volume (µL) and sperm concentration (nx10^7 per mL) after recovery by Method 1 Slicing+swim-up and Method 2, Mincing+flushing method
Method of sperm recovery
Volume of sperm recovered (µL)
Sperm Concentration (n x10
7 sperm per ml)
Slicing+Swim-up 500 ± 110b 123 ± 27.9
a
Mincing+flushing 770 ± 200b 189 ± 36.8
a
p value
0.28 0.18
Values in column with the same superscript do not differ by student’s T test (Statisitical significance P<0.05)
Table 3. Morphological characteristics of fresh epididymal sperm after using two methods of sperm recovery
224
Discussion
The ability to successfully recover viable epididymal sperm from post
mortem testes constitutes the initial step in germplasm conservation of
genetically diverse native animals for future utilization using assisted
reproductive biotechniques. Optimizing a procedure that will yield good
quality epididymal spermatozoa from post mortem testes is a key element when
one is to engage in basic and applied research relative to sperm
cryopreservation of indigenous/native goats in the Philippines for future
Animal Genetic Resources (AnGR’s) cryobanking.
In this study, two methods of epididymal sperm recovery were
compared to determine the characteristics of sperm that can be recovered. A
larger volume of spermatozoa can be isolated in mincing+flushing
(Method 2) because of the greater surface area to be exposed allowing
greater number of spermatozoa to be flushed. However, the presence of
epithelial cells and blood isolates in such procedure can not be avoided
primarily due to the cut capillary vessels releasing hemolyzed blood cell
contents. Furthermore, the likelihood of getting morphologically abnormal
sperm eg. detached head and dead sperm is increased due the mechanical
method of isolation causing a larger number of retrievable sperms.
In the slicing and swim-up or method 1, the volume of sperm was
slightly lower because sperms gradually released themselves freely into the
Tris buffer solution only from the sliced portion of the cauda epididymis.
Such procedure produced a pale white turbid appearance with no traceable
tinge of hemolyzed blood. As a result, there is lesser chance of getting
morphologically injured, abnormal or dead cells. The slice+ swim-up